The potential of digitoxin as a cancer therapeutic has been extensively investigated; however a narrow therapeutic index and potential for toxic side effects have precluded its use in clinical settings as an anti-cancer agent. The long-term goal of this study is to develop, using a combinatorial rational drug-design approach, novel analogues of digitoxin that possess enhanced anti-tumor activity against lung cancer as compared to existing cardiac glycosides but with lower side effects. This study is directly relevant to the mission of the NHLBI. These analogues will be generated using a divergent de novo palladium-catalyzed carbohydrate synthesis methodology and tested using in vitro, ex vivo and in silico techniques. Further, important apoptotic and angiogenic mediators that are critical to the anti-tumor effect of digitoxin and its analogues in lung cancer will be delineated. Aim I is designed to characterize the potent apoptotic effect induced by an already identified novel monosaccharide analogue of digitoxin (MonoD), identify key intracellular regulators of its pro-apoptotic effect, ad delineate the role of free radicals that mediate MonoD and digitoxin action on lung cancer cells. Preliminary data clearly indicates that MonoD, synthesized using the palladium-catalyzed approach, demonstrates more than five-fold higher cytotoxic activity than digitoxin on NCI's panel of sixty cancer cell lines, and is particularly potent against NSCLC cell lines. Further, dat shows that the anti- proliferative effects of MonoD may be mediated by modulation of Bcl2 and caspases, which will be evaluated here. In Aim II, the tumor-angiostatic potential of MonoD and digitoxin, the underlying mechanisms of action, and the role of free radicals will be investigated. This is supported by preliminary data showing potent anti- angiogenic potential for MonoD, and inhibition of the key angiogenic mediators Akt and HIF1?. The objectives of this aim will be achieved using both in vitro and ex vivo techniques, and potential cross-talk between the apoptotic (Bcl2) and angiogenic (HIF1?, VEGF) pathways will be investigated. To further improve upon the efficacy and specificity of MonoD and digitoxin and identify novel biding substrates for these drugs, a rational drug-design approach is proposed in Aim III. As opposed to traditional SAR techniques, homology modeling and docking, in silico techniques that are quicker and more efficient will be employed. In addition, binding studies will be used to identify novel binding substrates for MonoD and digitoxin. Upon identification of suitable potential drug derivatives of digitoxin and MonoD, the inherently flexible de novo methodology will be used to synthesize the new candidate drugs. These drugs will be validated for specificity of action using techniques in Aim I and Aim II, and validated for anti-tumor activity by measuring proliferation, migration, invasion and tumor-metastasis. This study is important not only for the understanding of the mode of action of cardiac glycosides in lung cancer cells, but also to generate a new class of drugs that may target multiple novel pathways critical to tumor progression, and thus may prove to be effective across a variety of cancers.